mdm2 RNA splice variants in ovarian cancer identified by RT-PCR and DNA sequencing. The table shows sizes of splice variants, splice sites and tissues in which the splice variant was identified. (First nucleotide of coding sequence is #312, last nucleotide of coding sequence is #1784). The length of the splice variants includes only the coding sequence but not the flanking primer sequence. (> indicates exon/intron boundary and < indicates intron/exon boundary)

Abbildungsverzeichnis

Structural and functional regions of the p53 protein (redrawn from May and May, 1999) Functional regions and corresponding amino acid residues are shown on top. In the middle, evolutionary highly conserved domains I-V are shown. The bottom represents the tertiary structure of the site-specific DNA-binding. L1, L2, L3 indicate loops, and LSH indicates a loop-sheet-helix structure. The tertiary structure is shown in more detail in Fig. 2.

Topological diagram of the secondary structure elements of the core domain of the p53 protein (redrawn from Cho et al. 1994). The residues at the start and the end of each secondary structure element are indicated. The DNA-binding regions of the protein (L1-S2-S‘, L2, L3, S10-H2) correspond to the conserved regions of the p53 gene and are colored yellow for region II (residue 117-142), red for region III (residue 171-181), blue for region IV (residue 234-258) and green for region V (residue 270-286). The boundaries of the two ß-sheets that make up the ß-sandwich are shaded. The scaffolding regions are white.

p53 pathways. Upstream activators are shown above in red. Multiple downstream effectors of p53 which play a role for cell cycle arrest and apoptosis are shown. Arrows indicate a positive effect, while a flat line indicates inhibition. Quadrangles indicate specific functions in p53/MDM2 interaction.

Hypothetic model about the effects of impaired MDM2 function on p53 protein accumulation in the cell. Cellular stresses cause p53 activation, which is downregulated by functional MDM2 in an autoregulatory feedback loop. In case of nonfunctional MDM2, wildtype p53 accumulates in the nucleus.

mdm2 splice variant cDNA after NcoI restriction enzyme digestion of the pcDNA3 expression vector. The expression vector has three NcoI restriction sites at positions 610, 1976, and 2711 and the mdm2 cDNA (654 bp) has one NcoI restriction site at position 533. The cDNA analyzed on the agarose gel shows three fragments of the vector and two fragments of the cDNA insert, one with additional vector sequence. All clones (lanes 1-6) contain the mdm2 splice variant cDNA.

Transient transfection of pcDNA constructs into vaccinia virus infected HeLa cells. The cDNA of interest was inserted into the pcDNA3 plasmid such that it came under the control of the T7 RNA polymerase promoter (pT7). Using liposome-mediated transfection, this recombinant plasmid is introduced into the cytoplasm of HeLa-cells infected with the vTF7-3 strain, a recombinant vaccinia virus encoding bacteriophage T7 RNA polymerase. During incubation, the cDNA is transcribed with high efficiency by T7 RNA polymerase. Proteins were detected by immunoprecipitation and Western blotting.

p53 mutations in evolutionary highly conserved domains of the gene. The upper part of the figure shows the number of mutations in codons 36 through 324 of the p53 gene. Mutations in evolutionary highly conserved regions are marked in yellow, mutations in non-conserved regions in dark blue and splice site mutations in white. The lower part of the figure shows the corresponding DNA interacting regions of the p53 protein.

Intron 10 polymorphism of the p53 gene at nucleotide 17708
A novel polymorphism of the p53 gene was identified
at nucleotide 17708 (a>t) of intron 10 by SSCP analysi
and DNA sequencing in the ovarian cancer case #3402.

p53 protein overexpression according to type of p53 mutation. Overexpression is found in a high percentage of cases with missense mutations,
while the frequency of overexpression in nonmissense mutations is overall only 46%.

Southern hybridization of genomic DNA with the mdm2 cDNA probe. The sarcoma cell line SA1 which was used as a positive control shows amplification of mdm2 DNA, while none of the ovarian cancer tissues shows DNA amplification.

Northern hybridization of total mRNA with the mdm2 cDNA probe. The sarcoma cell line SA1 which was used as a positive control shows overexpression ofmdm2 RNA, while none of the ovarian cancer tissues shows mdm2 overexpression.

mdm2 RNA splice variants in ovarian carcinomas. Total RNA was ana-lyzed by RT-PCR, and PCR-products were separated on a 1.2% agarose gel. Cases
in lanes 1,2,6,7,9, and 10 show splice variants of different sizes. The case in lane 3
shows no mdm2 expression. (cDNA bands on the gel contain additional 53 bp of flanking primer sequence)

A-B: RT-PCR for ß2 microglobulin as a positive control for quality and amount of RNA.(A) RNA was extracted from normal ovarian tissue. All cases show
equal amplification of a 898 bp ß2M PCR product. Lane 21 shows the PCR product of
cDNA from the SA1 sarcoma cell line. Lane 22 is the negative control. (B) RT-PCR
for ß2 microglobulin expression in ovarian cancer RNA. Except for case #2341 in lane
7 all cases show approximately equal expression of the ß2 microglobulin RNA.

Loss of functional regions in mdm2 splice variants in comparison to the full length mdm2 gene Above, functional domains of the MDM2 protein with corresponding amino acid residues are shown (according to Freedman et al. 1999). Below, splice variants of the mdm2 gene are shown. Black lines indicate the missing part of the sequence.

A-D: Sequence overlaps in mdm2 RNA splice variants. (A-C) Splice site sequences of the 391 bp and 221 bp mdm2 splice variants show overlaps of several basepairs. The capital letters refer to transcribed bases that remain in the final mRNA products, while the lower case letters refer to untranscribed bases which are spliced out of the final mRNA product. The green boxes enclose the transcribed sequence at the donor site and the blue boxes enclose the transcribed sequence at the acceptor site. The yellow boxes indicate sequence homologies. D) As opposed to many of the aberrant splice variants which show overlapping sequences at the splice site, no such sequence homology is notable in the 654 bp mdm2-b splice variant which splices at exact exon/intron boundaries of exon 3 and exon 12.

mdm2 RNA splice variants in ovarian cystadenomas and tumors of borderline malignancy. Total RNA was analyzed by RT-PCR and PCR-products were
separated on a 1.2% agarose gel. Cases in lanes 1, 2, 5-8, and 10 show splice variants
of different sizes. The case in lane 3 shows no mdm2 expression despite normal
expression of the ß2-microglobulin control gene. (cDNA bands on the gel contain additional 53 bp of flanking primer sequence).

mdm2 RNA splice variants in normal ovarian tissue. Total RNA of ovarian tissue was analyzed by RT-PCR and PCR-products were separated on a 1.2%
agarose gel. Cases in lanes 3, 5, 7, and 8 show splice variants of different sizes. (cDNA bands on the gel contain additional 53 bp of flanking primer sequence).

Cytoplasmic expression of p53 and MDM2 proteins by transient transfection of pcDNA3 vector constructs into vaccinia virus infected HeLa cells A: Expression of the full length p53 protein (53 kDa). B: Expression of the full length MDM2 protein (90 kDa).C: Expression of a 40 kDa protein of the splice variant mdm2-b (654 bp). No protein could be expressed from the mdm2 splice variants of 351 bp and 52 bp. Proteins were analyzed by Western blotting and anti-myc respectively anti-HA immunodetection.

Clinical outcome of ovarian cancer patients dependent on mdm2 RNA alternative splicing (n=92) as analyzed by RT-PCR and DNA sequencing. C) Overall survival in patients with expression of small splice variants of <300 bp versus expression of the mdm2-b splice variant (654 bp) or full length mdm2D) Overall survival in patients with expression of small splice variants (<300 bp) versus expression of mdm2 splice variants of >300 bp in the presence or absence of the full length mdm2 transcript. Cases with expression of the the mdm2-b splice variant (654 bp) were not included in this analysis.

Clinical outcome of ovarian cancer patients dependent on mdm2 RNA alternative splicing (n=92) as analyzed by RT-PCR and DNA sequencing. E) Overall survival in patients with expression of the mdm2-b splice variant (654 bp) versus absence of the mdm2-b splice variant.F) Overall survival in patients with expression of a small splice variant of 221 bp was correlated with early stage (FIGO I/II) and marginally significant with a better clinical outcome.